Acquity system update - Waters Corporation · 2013. 9. 10. · ©2013 Waters Corporation 4 ACQUITY...

©2013 Waters Corporation 1

Acquity system update

Tony Wiklund

Nordic application chemist


Outline

Acquity I-Class

Acquity UPC2

Acquity APC

Acquity Masstrak online SPE analyzer


Extending the ACQUITY UPLC Family…


ACQUITY UPLC I-Class System What is it?

Binary solvent manager (BSM)

– 1200 bars

– New flow path with smaller ID

Sample manager (SM-FTN or SM-FL)

— Less dispersion volume (tubing, valve)

— Improved carry over

Column ovens

— Single or multiple column

— New APH with smaller ID

New cells for TUV and PDA

— 500 nl/10mm

— 250 nl/10 mm (for 1mm ID columns)

Compatible with FLR, ELSD & all MS

Full range of ACQUITY UPLC Column chemistries


Instrument Contribution to Bandspread/Extra-Column Effects

Engineering developments have specifically improved dispersion

– Injector design, injector volume, fittings, flow path, sealing surfaces

– Reduced tubing volumes - higher pressure/extended flow rate range

– Improved flow cell dispersion

22

det

2

,det

2

,

2

,

2

,

2

,

2

, Fectorectorvpostcolumnvcolumnvprecolumnvinjectorvtotalv

Injection volume

+ injector band-

spreading

Tubing between injector

and column

Column volume

+ frits

Tubing between column

and detector

Band- spreading inside the detector

cell +

tubing

Time-based Band-

spreading in the

Detector (Sampling Rate; Time Constant)


Tubing ID Volume/foot Volume/100 cm

0.009’’ 12 µl 42 µl

0.005’’ 3.5 µl 12.5 µl

0.004’’ 2.5 µl 8.2 µl

0.003’’ 1.4 µl 4.6 µl

How does it translate to chromatographic performances?

– Peak capacity is increased (with decreasing ID)

– Peak shape is improved

– Overal chromatographic performances are strongly impacted

– Pressure drop created with tubings is increased

o P 1/ID4

0.003’’ vs 0.004’’: 50% less volume, 3x more pressure

Higher pressure limit is required, just a tool, a technology enabler

Higher pressure is not a goal

Extra Colum Volume Contribution Of Tubing ID


Two NEW Sample Managers

Fixed Loop

New low dispersion fittings, lower dispersion

needle seal for the FTN

Optional extension coil

System volume <100 µL

Flow Through Needle

New low dispersion fittings, shorter sample

path for the FL (10 µL)

Low dispersion 1, 2, 5 & 10-µL loop design

— Conventional 20, 100, and

250 µL available

System volume <95 µL

New EverFlow inject valve design

to enable higher pressures

ACQUITY UPLC H-Class chassis and robust

rotary sample tray/plate mechanism

Compatible with newest Sample Organizer (18

shelves)

Time0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88

%

0

100

0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88

%

0

100

Omeprazole 10ng 0403 MRM of 1 Channel ES+ 346.083 > 198.068 (Omeprazole)

7.06e7

Blank1 10ng 0403 MRM of 1 Channel ES+ 346.083 > 198.068 (Omeprazole)

4.42e3

0.103

0.136

0.205

0.249

0.276

0.294

0.308ps

i

13500.00

14400.00

15300.00

16200.00

17100.00

18000.00

Minutes0.00 0.15 0.30 0.45 0.60 0.75


I-Class FTN vs. I-Class FL: Comparison Summary

ACQUITY I-Class FTN I-Class FL

Dwell Volume* 120 µL 100 µL 95 µL

Bandspread* 12 µL <9 µL <7 µL

Carryover 0.005 % 0.001 % 0.002 %

Precision <0.3% Full loop <1.0% PLUNO

<1% 0.2 to 1.9 μL <0.5% 2 to 10 μL

<0.3% Full loop <1.0% PLUNO

Cycle Time < 15 sec

(with load ahead) <30 sec

< 15 sec (with load ahead)

User Simplicity 3 injection modes

loop changes Single injection

mode 3 injection modes

loop changes

*Measured with a complete system – Measurement on module only doesn’t make sense


Column Ovens

NEW Column Heater

Two (2) Columns plumbed right or

left only

New 0.003”/0.075 mm I.D. tubing

Robust 18 K psi fitting

For method development or multi-

method support

NEW Column Manager

New 0.003”/0.075 mm I.D. tubing

Robust 18 K psi fitting

Excellent method transfer between

ACQUITY UPLC I-Class Systems

CH-30A is compatible where HPLC

columns must be supported


Accelerate Ballistic Separations ACQUITY UPLC and ACQUITY UPLC I-Class – 1 mm I.D. column

Instrument Method Name: 30sec_10_95p600mL_90C ACQUITY

0.1

58

0.2

05

0.3

51

0.4

19

0.4

57

0.4

83

0.5

03

AU

0.000

0.022

0.044

0.066

0.088

0.110

Instrument Method Name: 20sec_NO hold 10_95_p904mL_90C

0.0

98

0.1

17

0.1

70

0.2

13

0.2

41 0.2

61

0.2

76

AU

0.000

0.022

0.044

0.066

0.088

0.110

Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

Average peak capacity at 5σ = 57

Average peak capacity at 5σ = 70

ACQUITY UPLC

ACQUITY UPLC I-Class

Enhanced backpressure limits allows the use of optimum flow rates for 2.1 and 1.0 mm I.D. columns packed with 1.7 m particles resulting in best speed and resolution.


I-Class


The UPLC Family


Outline

Acquity I-Class

Acquity UPC2 (Guy Wilson, G10)

Acquity APC



UPC2 UltraPerformance Convergence Chromatography

Expanding Selectivity for the

Chromatographic Laboratory


Evolution of Separation Technology

Gas Chromatography Liquid Chromatography Convergence Chromatography

GC

Capillary GC

HPLC

UPLC

SFC

UPC2


UltraPerformance Convergence Chromatography

Convergence Chromatography is a category of separation science that

provides orthogonal and increased separation power, compared to liquid or

gas chromatography, to solve separation challenges.

UltraPerformance Convergence Chromatography [UPC2] is a

holistically designed chromatographic system that utilizes liquid CO2 as a

mobile phase to leverage the chromatographic principles and selectivity of

normal phase chromatography while providing the ease-of-use of reversed-

phase LC.

The ACQUITY UPC2 System is built utilizing proven UPLC Technology to

enable scientists the ability to address routine and complex separation

challenges while delivering reliability, robustness, sensitivity and throughput

never before possible for this analytical technique.


Sample Preparation Major Source of Laboratory Costs

Sample preparation is the most often cited area of improvement to save time and operating costs

Most sample preparation involves being in an organic phase

– Liquid/Liquid, PPT, Soxhlet, Distillation, Evaporation and Reconstitution

Many matrices will respond best to organic phases (gels, blisters, ointments, synthesis solvents, etc.)

image from dyapharma.com image from sefetec.net image from tasnee.com


Improving Workflow with Convergence Chromatography

SPE Extraction (Florisil)

Gas Chromatography

STEP 1

Elution in hexane/ethyl acetate

STEP 2

Evaporate to dryness STEP 3

Reconstitute in cyclohexane

STEP 4

pyrethroids

Derivatize sample

Ready for analysis

STEP 5

STEP 6

SPE Extraction (Oasis HLB)

Reversed-phase LC

Elution in methanol

Evaporate to dryness

Reconstitute in water

carbamates

Ready for analysis

Convergence Chromatography

Direct Analysis on UPC2


Eliminate lengthy evaporation and

reconstitution steps

No need for derivatization


Separation Technology Overview

Gas Chromatography

Liquid Chromatography


Separation achieved by a temperature gradient •High efficiency [N]

• Virtually no limitation on column length •Limited selectivity [α]

• Limited stationary phase options

Separation achieved by a solvent gradient •High efficiency [N]

• Limited to pressure drop across column

• Moderate selectivity [α] • Different modes: reversed-phase, normal-phase, SEC, IEX, affinity, ion pair, HILIC, GPC…etc.

Separation achieved by density/solvent gradient •High efficiency [N]

• Very low viscosity enables longer columns and smaller particles •High selectivity [α]

• Wide variety of stationary phase and mobile phase co-solvent and modifier options

GC

LC

CC


The Advent of Convergence Chromatography


SFC UltraPerformance Convergence Chromatography

Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland

UltraPerformance Convergence Chromatography is the result of significant technological advancements in Supercritical Fluid Chromatography instrumentation and chemistry design while providing exceptional increases in available selectivity


Applicability of UPC2: Fast Chiral Screening

Fast Chiral Screening

Key advantages of moving to UPC2

– Results that are equal to or better

– 30X reduction in analysis time

– Nearly 75X reduction in solvent

• 135 µL of MeOH vs 10 mL of hexane/ethanol

Providing meaningful impact to scientists from

discovery to QC based on the reducing of non-value

adding steps in analytical workflow process

Reduces the time consuming solvent mixing and

sample preparation so can reallocate resources to

other value adding analytical work

Increases the column lifetime so can reallocate

consumable budget

Reduces the cost of solvent investments of purchase

and removal

Reduces the complexity of instrument multi-method

use so can reduce the capital investments, or increase

value added human resources

AU

0.00

0.12

0.24

0.36

0.48

Minutes

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

AU

0.00

0.30

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

UPC²

NPLC 11 min

0.3 min


Applicability of UPC2: Normal Phase Replacement

6.2

37

10.8

55

20.8

50

26.6

32

30.8

56

35.8

19

AU

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

ACQUITY UPC² 15 min Cost per run ~ $0.05

Normal Phase HPLC 50 min Cost per run ~ $5.89

Normal Phase Replacement

Providing meaningful impact to scientists from

discovery to QC based on the reducing of non-value

adding steps in analytical workflow process

Reduces the time consuming solvent mixing and

sample preparation so can reallocate resources to

other value adding analytical work

Increases the column lifetime so can reallocate

consumable budget

Reduces the cost of solvent investments of purchase

and removal

Reduces the complexity of instrument multi-method

use so can reduce the capital investments, or

increase value added human resources

Key advantages of moving to UPC2

Results that are equal to or better

Over 3X reduction in analysis time

Cost per analysis just from solvent use from $5.89

to 5 cents


Diverse Applicability of Convergence Chromatography

A 4-hydroxyacetophenone B p-nitrophenol C 2-hydroxyacetophenone D 4-chloroacetanilide E acetanilide F phenacetin G 3-aminophenol H paracetamol I 2-aminophenol

Pharmaceutical Impurity Profile

Environmental Food and Beverage

Chemical Materials

Non-ionic surfactants Paracetamol and impurities

Chiral pesticides Vitamin E isomers

Permethrin


Built upon proven UPLC Technology

– Quantifiable increase in productivity

Exceptional increase in available selectivity

– Solve routine and complex separation

challenges


Outline

Acquity I-Class

Acquity UPC2

Acquity APC



A Novel, Practical Approach to

SEC/GPC Analysis

Introducing

ACQUITY®

Advanced Polymer Chromatography™

(APC™) System


What’s the Topic Today?

ACQUITY APC System

– Introduced at Pittcon 2013(Philadelphia)

GPC/SEC polymer characterization with

– sub-2µm /sub-3µm rigid hybrid particles

o BEH chemistry introduced in 2004

Complete solution

– New higher efficiency columns

o aqueous and organic

– New chromatographic system

o Designed for high efficiency columns

o Low dispersion, precise flow rate


Recent Trends Polymer Development

Green Chemistry - Decrease organic solvents in

processes

- Increase water based processes

- Bio-sourced polymers

- Biodegradable polymers

Key: Lower average molecular weight polymers

Modern Chemistry - Functional polymers

- End-groups - Pendant groups

- Better control

- Polymerization reactions - Molecular weight

averages & polydispersity

- Next gen catalysts

- New, innovative polymer structures


ACQUITY APC System

Analytical Challenges

ACQUITY APCTM System

Gel Based Columns

Typical materials include - Styrene-DVB - Methacrylates

Relatively fragile

Solvent to solvent conversion not easy

Speed of Analysis

Current approaches to size based separations compromise - Peak resolution

- Characterization

- Data quality

Poorly Resolved Low Molecular

Weight Polymer & Oligomers

Traditional GPC is a low resolution technique Low resolution limits characterization information

Innovative polymers and building blocks need high resolution


Limitations of High Speed Gel Permeation Chromatography

. Data Courtesy of Janco, M; Alexander, J; Bouvier, E; Morrison, D; Ultra High Performance Size Exclusion Chromatography of Synthetic Polymers; Pittcon 2013; Session 2360-1


Advanced Polymer Chromatography versus High Speed Gel Permeation Chromatography

Data Courtesy of Janco, M; Alexander, J; Bouvier, E; Morrison, D; Ultra High Performance Size Exclusion Chromatography of Synthetic Polymers; Pittcon 2013; Session 2360-1


Low MW Polymer: Resolution Realized

Polystyrene Standard 510 Mp

HPLC System with RI 6x150mm HSPgel HR1

ACQUITY APC System with RI 4.6x150mm; 45Å XT


Advanced Polymer Chromatography A Definition

Application technique for the size

based separation of polymers in

solution using columns packed with

sub-3µm, rigid, high-pore-

volume, hybrid particles combined

with a fully optimized low

dispersion ACQUITY system


Introducing the ACQUITY Advanced Polymer Chromatography (APC) System

Precise solvent

management

Low system dispersion

Compatibility with

challenging solvents

Rigid, solvent-resilient columns

Versatile column

management

Stable refractive

index detection

Flexible detection

techniques

Wide range of APC

standards


Speed of Analysis & Better Characterization

MV

0

5

10

15

20

25

30

35

Minutes

13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26

µR

IU

0

2

4

6

8

10

12

14

16

18

20

Minutes

2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.7

100K

10K

1K

100K

10

K

1K

GPC APC

3 x Styragel 7.8x300mm (4e, 2, 0.5)

3 x APC TMS 4.6x150mm (200,45,45)

µR

IU

0.0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

Minutes

3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70

MV

-1.0

0.0

1.0

2.0

3.0

4.0

Minutes 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0

1K polystyrene

standard 1K polystyrene

standard


More resolution = more

points for low molecular

weight calibration

Better calibration =

more accurate data

Lo

g M

ol W

t

2.40

2.80

3.20

3.60

4.00

4.40

4.80

5.2

5.6

Retention Time

15 16 17 18 19 20 21 22 23 24 25 26 27

Lo

g M

ol W

t

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

Retention Time

2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

Alliance GPC system

3 x Styragel 7.8 x 300mm (4e, 2, 0.5)

Polystyrene calibration (100K, 10K, 1K)

ACQUITY APC system

3 x APC TMS 4.6 x 150mm (200,45,45)

Polystyrene calibration (100K, 10K, 1K)

Speed of Analysis & Better Characterization


Gel Based Columns versus Rigid Hybrid Particles

THF

DMF

Toluene

One System. One Bank of Columns. Solvent Flexibility.


ACQUITY APC Column Options

Particles based on BEH chemistry

Five pore sizes – 45 Å (200 – 5,000) 1.7µm

– 125 Å (1,000 – 30,000) 2.5µm

– 200 Å (3,000 – 70,000) 2.5µm

– 450 Å (20,000 – 400,000) 2.5µm

– 900 Å* (TBD, later in the year)

Two surface chemistries – Organic solvent- XT

– Aqueous buffers – AQ

Three column lengths – 30 mm

– 75 mm

– 150 mm

Internal diameter: 4.6 mm


ACQUITY APC ISM Precision: 100 injections of Epoxy Resin Overlaid

µR

IU

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Minutes

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00

Injection Mp Mw Mn PDI 20 2743 6143 3870 1.587 40 2753 6160 3884 1.586

60 2754 6156 3884 1.585 80 2745 6146 3870 1.588

100 2746 6151 3878 1.586 % RSD 0.18 0.11 0.18 0.08

Long-term Repeatable Solvent

Delivery for Precise/Accurate

MW Determination

ACQUITY APC XT 4.6x150mm; 1 x 125Å (1,000-30,000) and 1 X 45Å (200-5,000)


Conclusion

New ACQUITY APC System

– Brings all the advantages of sub-2µm & sub-3µm particles to

polymer characterization (GPC/SEC)

Analysis time reduction by 5x (at minimum)

– Daily calibration is easy

New columns compatible with any solvent

– Rigid, BEH particles

– Use one column set for different solvents

Achieve better results faster

– Higher resolution, especially in low MW area


Outline

Acquity I-Class

Acquity UPC2

Acquity APC



MassTrak™ Online SPE Analyzer

Integrate. Accelerate. Deliver Better Results


Outline

The MassTrak Online SPE Analyzer

– The System

o Components and Capabilities

– Benefits of the System

o Increase Laboratory Productivity

o Better LC/MS Assay Results

o Faster, More Efficient Method Development

– Examples of the System in Operation

o Sample Clean Up

o Method Development

o Specific Analytes


MassTrak Online SPE Analyzer Description

MassTrak Online SPE Analyzer

– Utilizes advanced fluidics and SPE Cartridge technology to integrate

SPE Sample Preparation and LC/MS

Analyzer Components:

o ACQUITY® UPLC Sample Manager

o ACQUITY UPLC Binary Solvent Manager

o ACQUITY UPLC Column Manager – A

o ACQUITY Online SPE Manager (OSM)

o ACQUITY TQ Detector (or Xevo™ TQ or TQS)

o MassTrak SPE Cartridges

System controlled by MassLynx Software

Possible to retrofit OSM to existing LC/MS Systems

*NOTE: The MassTrak Online SPE Analyzer is under development


Components of the MassTrak Online SPE Analyzer

ACQUITY Sample Manager • Up to 48 Vials or 2 Microplate Temp controlled sample capacity • Up to 200 µl injection volume • Optional Sample Organizer expansion

ACQUITY Binary Solvent Manager • 4 solvent capacity

ACQUITY Column Manager • 4x 50mm column capacity

ACQUITY Online SPE Manager

Acquity TQ Detector

“A UPLC®-enabled online solid phase extraction system

ACQUITY TQ Detector


A Closer Look At The ACQUITY Online SPE Manager

Left Clamp (Elution)

Right Clamp (Loading)

High Pressure Dispenser SPE Solvent

Inlets

Valving to allow switching of modes

Gripper to move cartridges to & form clamps

2 x 96-well plates/trays of cartridges


Comparison of Online vs. Offline SPE Sample Preparation

Manual SPE OnLine SPE for HPLC

MassTrak Online SPE Analyzer

Manual Intervention Required

Required at Every Step

Automated Automated

Opportunity for Sample Processing Errors

Many throughout process

Minimal Minimal

Sample Preparation Consistency

Variable Very Good Very Good

User Exposure to Hazards

Repeated and Constant

Minimal Minimal


System Modes Of Operation

UPLC Mode

– Online SPE can be bypassed to allow UPLC operations

Sample Extraction Mode

– Online processing of samples by SPE integrated with LC/MS

Advanced Method Development Mode

– Method characterization (sample breakthrough, recovery & elution)

Multidimensional SPE Mode

– Use more than one type of SPE sorbent


Summary

The MassTrak Online SPE Analyzer

– Automates SPE Sample Preparation

– Integrates SPE Sample Preparation with LC/MS

System includes all components necessary for Sample

Preparation and Analysis

– ACQUITY UPLC

– ACQUITY Online SPE Manager

– ACQUITY TDQ MS System

– MassLynx Software


Summary

Use of the MassTrak Online SPE Analyzer offers several key benefits

– Increased Laboratory Productivity

o Better Use of Human and Instrument Resources

o Faster Time to Result

o Quick and Efficient Method Development

o Minimizes Opportunity for Sample Processing Errors and Variability

o Reduced Exposure to Chemical and Biological Hazards

– Improved LC/MS Assays

o Improved Sample Clean Up

o Increased Assay Sensitivity

o More Efficient and Robust Methods

– Appropriate for the Clinical Laboratory

o Intended for in vitro diagnostic use

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Acquity system update - Waters Corporation · 2013. 9. 10. · ©2013 Waters Corporation 4 ACQUITY...

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